Bacillus anthracis is the causative agent of anthrax, a zoonotic disease transmitted by spores that germinate in the infected host. The resulting vegetative forms of B. anthracis invade and replicate in all host organs, triggering a lethal outcome to infection and subsequently generating spores for dissemination. Formation of a poly-D-3-glutamic acid (PDGA) capsule represents a key virulence strategy of anthrax bacilli. Further, B. anthracis elaborates a protein surface (S)-layer that is composed of twenty-four S-layer proteins, which are required for this microbe's virulence. The functions of S-layer proteins include the adherence of bacilli to host tissues, the scavenging of heme-iron, and the control of B. anthracis chain length as an escape from phagocytic clearance. S-layer assembly is initiated via precursor transport across the bacterial plasma membrane by a specialized Sec machine. S-layer proteins subsequently associate via their S-layer homology (SLH) domains with pyruvylated / acetylated secondary cell wall polysaccharide (SCWP), a carbohydrate that is linked to peptidoglycan. S-layer proteins likely contribute to capsule assembly. In B. anthracis, linear PDGA strands are linked to peptidoglycan and threaded across the S-layer. Bacillus cereus G9241, which causes anthrax-like respiratory disease, elaborates a hyaluronic acid capsule; its genetic determinants are conserved in B. anthracis. We propose to use molecular genetic approaches and animal models of anthrax pathogenesis to identify key S-layer virulence factors of B. anthracis, to appreciate their molecular contribution to anthrax pathogenesis and to explore their potential for anthrax vaccine development. Our experimental plan entails studies on the chemical composition of the B. anthracis envelope, microscopic analysis of S-layer protein traffic and capsule synthesis, the structural biology of SLH domain association with SCWP, the characterization of the host receptor for B. anthracis and the development of conjugate capsule-S-layer protein vaccines to protect experimental animals against lethal anthrax challenge. PUBLIC HEALTH RELEVANCE: Efforts at generating a human anthrax vaccine from purified subunits, for example protective antigen (PA), have failed. Importantly, PA is dispensable for anthrax pathogenesis. An important frontier is the identification of B. anthracis antigen(s) essential for virulence and that, when used as immunogens, elicit anthrax protective immunity. These questions are addressed in our proposal.